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. 2010 Jul;192(13):3304-10.
doi: 10.1128/JB.00172-10. Epub 2010 Apr 23.

Methanococci use the diaminopimelate aminotransferase (DapL) pathway for lysine biosynthesis

Yuchen Liu  1 , Robert H White, William B Whitman
Affiliations

Methanococci use the diaminopimelate aminotransferase (DapL) pathway for lysine biosynthesis

Yuchen Liu et al. J Bacteriol. 2010 Jul.

Abstract

The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to ll-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapL1 group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Deltammp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using ll-DAP and alpha-ketoglutarate as substrates was 24.3 + or - 2.0 nmol min(-1) mg of protein(-1). The gene encoding the DapL homolog in Methanocaldococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70 degrees C and pH 8.0 to 9.0. The apparent K(m)s of MJ1391 for ll-DAP and alpha-ketoglutarate were 82.8 + or - 10 microM and 0.42 + or - 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl-DAP as a substrate. Phylogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales.

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Figures

FIG. 1.
FIG. 1.
Variations in the DAP pathway for lysine biosynthesis. 1, succinylase pathway; 2, acetylase pathway; 3, aminotransferase pathway; 4, dehydrogenase pathway. Abbreviations and designations: THDPA, l-2,3,4,5-tetrahydrodipicolinate; l,l-DAP, ll-2,6-diaminopimelate; meso-DAP, meso-2,6-diaminopimelate; LysC, aspartate kinase; Asd, aspartate semialdehyde dehydrogenase; DapA, dihydrodipicolinate synthase; DapB, dihydrodipicolinate reductase; DapD, THDPA succinylase; DapC, succinyl-DAP aminotransferase; DapE, succinyl-DAP desuccinylase; DapF, DAP epimerase; LysA, DAP decarboxylase; DapL, ll-DAP aminotransferase; Ddh, DAP dehydrogenase.
FIG. 2.
FIG. 2.
Growth requirement of the Δmmp1527::pac mutant S600 for lysine. (A) Growth in McNA. (B) Growth in McNA containing lysine (1 mM). •, wild-type strain S2; ○, Δmmp1527 mutant S600; ▴, strain S601 (complemented strain S600 with mmp1527 expressed from pMEV2-Mmp1527). The growth curves are representative curves for experiments that were independently replicated three times. All values are the averages of triplicate cultures.
FIG. 3.
FIG. 3.
DapL activity (A) and heat stability (B) of MJ1391. (A) The DapL assay was performed at 70°C for 0 to 60 min before termination and measurement of the product glutamate. (B) The temperature stabilities of MJ1391 and the substrates α-KG and ll-DAP were examined by preincubation of the MJ1391 protein (open bars), the substrates (gray bars), or the protein and the substrates (incubated separately) (black bars) at 70 to 100°C in Tris-HCl buffer (pH 8.5) for 30 min before the DapL activity assay was performed at 70°C. One hundred percent activity was defined as the activity that was observed without preincubation (0.30 μmol min−1 mg of protein−1). The error bars indicate the standard deviations of three assays.
FIG. 4.
FIG. 4.
Optimum temperature (A) and optimum pH (B) for MJ1391. Assays were conducted for 30 min. One hundred percent activity was defined as the activity that was observed at 70°C and pH 8.5 (0.33 μmol min−1 mg of protein−1). The assays were conducted at pH 8.5 (A) and at 70°C (B).
FIG. 5.
FIG. 5.
Phylogenetic analyses of the DapL homologs. Red, bacterial DapL1; blue, bacterial DapL2; orange, archaeal DapL1; green, archaeal DapL2; cyan, methanococcal DapL. (A) Phylogenetic tree constructed by the minimum evolution method using the MEGA4 program. Scale bar = 0.1 amino acid substitution per site. Organisms whose enzymes have biochemically confirmed DapL activity are indicated by an asterisk (18, 24). Filled circles at branch points indicate ≥70% replication for 1,000 bootstraps. The grouping of DapL1 and DapL2 is based on the findings of Hudson et al. (18). Several aminotransferases from E. coli, B. subtilis, Bordetella parapertussis, and C. glutamicum, which are organisms that are known to lack DapL activity, were included to provide an overall context. The locus tags of the sequences used in the phylogenetic analysis are as follows: Protochlamydia, pc0685; Chlamydia, CT390; E. coli 1, b2379; E. coli 2, b3359; Gloeobacter, glr4108; B. subtilis 1, BSU37690; B. subtilis 2, BSU13580; B. subtilis 3, BSU11220; B. subtilis 4, BSU03900; Bacteroides, BF2666; Bordetella 1, BPP2543; Bordetella 2, BPP1996; Synechocystis, sll0480; Leptospira, LIC12841; Methanococcoides, Mbur1013; Methanosarcina barkeri, Mbar_A2605; Methanosphaera, Msp0924; Methanospirillum, Mhun2943; Moorella, Moth0889; Archaeoglobus, AF0409; Methanosarcina acetivorans, MA1712; Methanosarcina mazei, MM2649; Methanosaeta, Mthe0801; Methanothermobacter, MTH52; Corynebacterium 1, NCgl1343; Corynebacterium 2, NCgl1058; Desulfitobacterium, Dhaf1761; Arabidopsis, AT4G33680; Syntrophobacter, Sfum0054; Methanocorpusculum, Mlab0633; Methanoculleus, Memar1915; Methanobrevibacter, Msm1455; methanogen RCI, RCIX1962; Methanoregula, Mboo2096; Methanococcus aeolicus, Maeo1494; Methanococcus vannielii, Mevan0840; Methanococcus maripaludis, MMP1527; Methanocaldococcus jannaschii, MJ1391; and Methanococcus voltae, Mvol0315. (B) RED plot of DapL homologs. Intradomain comparisons within each DapL subgroup are indicated by diamonds that are a single color. Comparisons of different subgroups are indicated by diamonds that are two colors. (C) Hypothetical gene tree illustrating the proposed evolutionary history of DapL homologs. The solid lines show the organismal phylogeny in the absence of LGT. The dashed lines show the gene tree for DapL. T1 indicates the acquisition of bacterial DapL1 by archaea, and T2 indicates the acquisition of archaeal DapL2 by bacteria.
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